Hepatitis C virus (HCV) remains a major threat to public health despite the advent of specific inhibitors targeting viral protease and RNA polymerase. Resistance development, as a natural outcome from attack by therapeutics that directly kill or inhibit the growth of virus, demands alternative and more powerful therapeutics. Our laboratory has been studying the mechanisms of HCV entry in the hopes of identifying novel cellular targets for antiviral development. Entry inhibitors targeting host functions will be particularly valuable in treating post-transplant hepatitis C, where one of the major unmet needs is the development of strategies to prevent re-infection of the liver graft after transplantation. e and others previously demonstrated that tight junction protein occludin is a novel HCV entry factor. During the previous funding period, we then went on to elucidate additional human factors required for viral entry into cells, and have now acquired the first complete map of the molecular interactions between HCV glycoprotein E2 and the host. Our findings show that two host scaffold proteins, prohibitin 1 and 2, regulate HCV entry at a post- binding step through their interactions with CRaf. This achievement opens unprecedented opportunities to dissect the HCV entry process and to translate basic research into antiviral development. In this competing renewal, we propose to dissect the master signaling pathway that coordinates HCV entry. Our studies will, for the first time, define the role of the prohibitin-CRaf signaling pathway in HCV entry, elucidate the molecular events governed by prohibitin-CRaf activation, and explore the possibility of targeting prohibitins as a way to stop HCV infection. Taken together, accomplishing the project will significantly advance our understanding of HCV entry, and potentially lead to a new class of entry inhibitors.